Lack of detection of human immunodeficiency virus type 1 in the saliva of infected children and adolescents.

OBJECTIVE: To determine the prevalence of human immunodeficiency virus type 1 (HIV-1) in the saliva of infected children and adolescents. METHODS: Saliva and blood samples were collected from 13 patients (age range, 1-15 years) with HIV-1 infection. Eleven were taking antiretroviral agents. The presence of HIV-1 was determined by polymerase chain reaction analysis of RNA and DNA as well as by viral culture of the saliva samples and by culture of peripheral blood mononuclear cells. RESULTS: Although HIV-1 was cultured from peripheral blood mononuclear cells of 12 patients, it was not cultured from their saliva. Only 1 of 13 saliva samples yielded positive test results for HIV-1 RNA, and none did so for HIV-1 DNA. The specimen containing HIV-1 RNA was from an untreated 10-year-old asymptomatic boy with a CD4+ lymphocyte count of 0.91 x 10(9)/L (913 cells/microL) and no infectious virus detected in plasma. CONCLUSION: The prevalence of HIV-1 in the saliva of HIV-1-infected children and adolescents is low and may not be infectious.

Herpes simplex virus type 2 detection by culture and polymerase chain reaction and relationship to genital symptoms and cervical antibody status during the third trimester of pregnancy.

OBJECTIVES: Our goal was to define the frequency of asymptomatic herpes simplex virus type 2 shedding by culture and polymerase chain reaction and to correlate our findings with cervical anti-herpes simplex virus type 2 immunoglobulin A production. STUDY DESIGN: Women who were seropositive for herpes simplex virus type 2 collected daily genital tract samples during the third trimester for culture and deoxyribonucleic acid quantitation by polymerase chain reaction. Cervical secretions were collected weekly for anti-herpes simplex virus type 2 immunoglobulin A. Asymptomatic shedding by culture versus polymerase chain reaction and anti-herpes simplex virus type 2 immunoglobulin A detection with and without genital shedding were compared by means of McNemar's chi 2 test. RESULTS: Asymptomatic shedding was more frequent by polymerase chain reaction than by culture (13.8% vs 2.3%, p < 0.0001). When cervical anti-herpes simplex virus type 2 immunoglobulin A was present, patients were more likely to have negative results by polymerase chain reaction than positive results (66.7% vs 26.7%, p = 0.001). Anti-herpes simplex virus type 2 immunoglobulin A was detected beyond 37 weeks in only one subject. CONCLUSIONS: Polymerase chain reaction was more sensitive than culture for detecting asymptomatic genital herpes simplex virus. The role of immunoglobulin A in clearing genital herpes simplex virus remains to be determined.

Frequent detection of genital herpes simplex virus DNA by polymerase chain reaction among pregnant women.

OBJECTIVE: To investigate the prevalence and level of genital herpes simplex virus (HSV) among women at delivery. DESIGN, PATIENTS, AND SETTING: A prospective analysis of HSV by culture and by polymerase chain reaction (PCR) of genital specimens and by HSV serologic studies in 100 asymptomatic women in labor; prospective analysis of HSV by PCR among 50 seronegative nonpregnant women at a student health center; and retrospective analysis of genital specimens for HSV by PCR from 17 HSV culture-positive women with uninfected neonates and from two HSV culture-negative women with HSV-infected neonates. All pregnant women were at a university hospital. MAIN OUTCOME MEASURES: Presence of HSV by culture and levels of HSV by quantitative, type-specific PCR in cervical and vulvar specimens; HSV serologic testing by Western blot. RESULTS: All of the 100 asymptomatic women in labor who were studied prospectively were HSV culture negative. In nine HSV was recovered by PCR. Herpes simplex virus was recovered by PCR in one of the 50 seronegative nonpregnant women; she soon became seropositive. All 17 culture-positive women had HSV recovered by PCR. High levels of HSV DNA were obtained by PCR from the two culture-negative women with infected neonates. Among those from whom HSV was recovered by PCR, HSV DNA levels were 250 times higher from culture-positive samples than from culture-negative samples (11,571 genome equivalents vs 46 genome equivalents; P < .001). CONCLUSIONS: The frequency of infant exposure to HSV DNA-containing secretions from HSV-seropositive mothers is about eight times higher than previously reported using HSV culture methods. High maternal levels of HSV DNA may be associated with an increased frequency of transmission of HSV to the infant.

Herpes simplex virus detection from genital lesions: a comparative study using antigen detection (HerpChek) and culture.

The sensitivity of a rapid enzyme immunoassay test (HerpChek Direct Herpes Simplex Virus Antigen Test [DuPont Medical Products, Wilmington, Del.]) for the detection of herpes simplex virus (HSV) antigens in patient specimens was compared with HSV culture. HerpChek positivity for HSV occurred with 179 (65%) of 275 lesion specimens, whereas culture for HSV was positive for 145 (53%) lesions (P = 0.002). HerpChek was twice as sensitive as culture for the detection of HSV in late-stage lesions and was equivalent to culture for the detection of HSV in early lesions. We conclude that HerpChek provides greater sensitivity than culture for HSV detection in late-stage genital lesions.

Coamplified positive control detects inhibition of polymerase chain reactions.

Polymerase chain reactions (PCR) may fail to react because the substrate DNA is absent (true negative) or because of inhibition of specific amplification (false negative). The use of positive controls can increase confidence in negative PCR results by ruling out failure due to inhibition as a cause of the lack of amplification products. This report describes the construction and application of coamplified positive controls for herpes simplex virus and human herpesvirus 6 amplifications. Herpes simplex virus and human herpesvirus 6 PCR products were modified to generate control PCR products in which the original probe sequences were replaced by a Drosophila probe sequence. Implementation of the coamplified controls increased our specimen throughput in comparison with the parallel control amplifications used previously. Clinical laboratories using PCR for diagnosis of infectious diseases may find positive controls particularly helpful for increasing confidence that negative amplifications represent truly negative specimens.

Extended duration of herpes simplex virus DNA in genital lesions detected by the polymerase chain reaction.

To evaluate the utility of the polymerase chain reaction (PCR) for documenting herpes simplex virus (HSV) in persons with reactivated genital lesions viral isolation was compared with a recently developed PCR method. Three women experiencing four episodes of recurrent genital herpes were followed for 10 days per episode with daily examination and duplicate swabs of the lesions, one for HSV culture and one for PCR. HSV type 2 was cultured from three of four episodes and the mean duration of viral isolation from recurrent genital lesions was 2.6 days. PCR detected HSV DNA from lesion swabs during all four episodes, and HSV DNA was positive for an average of 6.8 days. HSV DNA was demonstrated in ulcerative lesions on 15 of 17 days versus 3 of 17 days by viral isolation (P less than .01). HSV PCR became negative when the lesions reepithelialized. These data suggest that PCR is a more sensitive measure of HSV infection than routine viral culture and that PCR detects the presence of HSV at times when culture is negative.

ATP-binding sites in the membrane components of histidine permease, a periplasmic transport system.

Two components of the histidine permease in Salmonella typhimurium, the membrane-bound P and M proteins, react with the photoaffinity labeling reagent 8-azido-ATP in isolated membranes. The extent of labeling is decreased by the addition of ATP and somewhat less by addition of GTP, CTP, UTP, and ADP. Cyclic AMP, NAD, FAD, and S-adenosylmethionine have little effect. We propose that one or both of these proteins have a site capable of binding an adenine nucleotide and that, therefore, they may be involved in the energy-coupling step in active transport.

Adenylate cyclase is required for chemotaxis to phosphotransferase system sugars by Escherichia coli.

We report that in Escherichia coli, chemotaxis to sugars transported by the phosphotransferase system is mediated by adenylate cyclase, the nucleotide cyclase linked to the phosphotransferase system. We conclude that adenylate cyclase is required in this chemotaxis pathway because mutations in the cyclase gene (cya) eliminate or impair the response to phosphotransferase system sugars, even though other components of the phosphotransferase system known to be required for the detection of these sugars are relatively unaffected by such mutations. Moreover, merely supplying the mutant bacteria with the products of this enzyme, cyclic AMP and cyclic GMP, does not restore the chemotactic response. Because a residual chemotactic response is observed in certain strains with residual cyclic GMP synthesis but no cyclic AMP synthesis, it appears that the guanylate cyclase activity rather than the adenylate cyclase activity of the enzyme may be required for chemotaxis to sugars transported by the phosphotransferase system. Mutations in the cyclic nucleotide phosphodiesterase gene, which increase the level of both cyclic AMP and cyclic GMP, also reduce chemotaxis to these sugars. Therefore, it appears that control of the level of a cyclic nucleotide is critical for the chemotactic response to phosphotransferase system sugars.

Involvement of cyclic GMP in intracellular signaling in the chemotactic response of Escherichia coli.

The intracellular signal that produces changes in swimming behavior when bacteria encounter attractants or repellents has not previously been identified. We suggest, based on the following lines of evidence, that cyclic GMP (cGMP) is involved in this signaling process in chemotaxis by Escherichia coli. (i) The addition of attractants to bacteria causes a transient increase in the intracellular level of cGMP, whereas a repellent stimulus decreases the level transiently. These changes do not generally occur in a mutant lacking chemotaxis-specific proteins. (ii) In the absence of chemoeffectors, both addition of cGMP to bacteria and reducing the intracellular cGMP level produce changes in swimming behavior, and a mutant with an abnormal swimming pattern has an altered intracellular cGMP level. (iii) cGMP modulates the demethylation reaction responsible for adaptation to stimuli. (iv) Mutants defective in components of the adaptation system have altered cGMP metabolism.

Attractants and repellents influence methylation and demethylation of methyl-accepting chemotaxis proteins in an extract of Escherichia coli.

During bacterial chemotaxis, attractants and repellents alter the methylation levels of the methyl-accepting chemotaxis proteins (MCPs). These methylation levels represent a balance between two enzymatic processes: methylation and demethylation. In vivo experiments previously have shown that chemoeffectors influence the demethylation process; effects on the methylation system have not been reported. Here we show that in a cell-free extract of Escherichia coli both methylation and demethylation of the MCPs are affected by attractants and repellents. Attractants enhance methylation and inhibit demethylation. Repellents inhibit methylation and stimulate demethylation. The cell-free system provides an opportunity for further study of the mechanisms by which attractants and repellents influence the levels of methylation of the MCPs.

Isolation, genetic analysis, and characterization of Escherichia coli mutants with defects in the lacY gene.

Five hundred thirty-five lacY mutants were isolated from an Escherichia coli strain carrying the lactose operon on an F' factor, either without mutagenesis or after mutagenesis with 2-aminopurine or N-methyl-N'-nitro-N-nitrosoguanidine. Crosses against 48 independently isolated deletions ending in the lacY gene divided the gene into 36 deletion groups. Suppressibility studies with 7 nonsense suppressor strains classified 276 mutants as nonsense mutants and 78 as missense (or nonsuppressible) mutants. One hundred seventy-nine mutants were "leaky" and could not be so allocated, and two were found to have small internal deletions. Nonsense mutants could in many cases be subdivided even within deletion groups on the basis of their suppressibility pattern, giving a total of 70 groups of nonsense mutants. Studies of these mutants allow the following conclusions: lactose and melibiose most probably do not have separate binding sites on the permease; the lacY region most likely consists of one cistron, and so both active transport and facilitated diffusion are functions of one protein; and finally, there is probably no small defined region of the permease responsible for energy coupling of transport. Furthermore, the strains and the analysis form the basis for a future functional study of the permease by biochemical techniques.

The synthesis of S-adenosylmethionine by mutants with defects in S-adenosylmethionine synthetase.

Some metK mutants of Salmonella typhimurium with constitutive methionine biosynthesis have no detectable S-adenosylmethionine (SAM) synthetase, the enzyme which converts methionine to SAM, the postulated corepressor of the methionine pathway. However, these mutants are not auxotrophic for SAM, an essential compound for many reactions.